Iron-sulphur cluster biogenesis via the SUF pathway.

Iron-sulphur (Fe-S) clusters are versatile cofactors, which are essential for key metabolic processes in cells, such as respiration and photosynthesis, and which may have also played a crucial role in establishing life on Earth. They can be found in almost all living organisms, from unicellular prokaryotes and archaea to multicellular animals and plants, and exist in diverse forms. This review focuses on the most ancient Fe-S cluster assembly system, the sulphur utilization factor (SUF) mechanism, which is crucial in bacteria for cell survival under stress conditions such as oxidation and iron starvation, and which is also present in the chloroplasts of green microalgae and plants, where it is responsible for plastidial Fe-S protein maturation. We explain the SUF Fe-S cluster assembly process, the proteins involved, their regulation and provide evolutionary insights. We specifically focus on examples from Fe-S cluster synthesis in the model organisms Escherichia coli and Arabidopsis thaliana and discuss in an in vivo context the assembly of the [FeFe]-hydrogenase H-cluster from Chlamydomonas reinhardtii.

Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production.

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]­hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H2 production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria.

Chalcogenide substitution in the [2Fe] cluster of [FeFe]-hydrogenases conserves high enzymatic activity.

[FeFe]-Hydrogenases efficiently catalyze the uptake and evolution of H2 due to the presence of an inorganic [6Fe-6S]-cofactor (H-cluster). This cofactor is comprised of a [4Fe-4S] cluster coupled to a unique [2Fe] cluster where the catalytic turnover of H2/H+ takes place. We herein report on the synthesis of a selenium substituted [2Fe] cluster [Fe2{µ(SeCH2)2NH}(CO)4(CN)2]2- (ADSe) and its successful in vitro integration into the native protein scaffold of [FeFe]-hydrogenases HydA1 from Chlamydomonas reinhardtii and CpI from Clostridium pasteurianum yielding fully active enzymes (HydA1-ADSe and CpI-ADSe). FT-IR spectroscopy and X-ray structure analysis confirmed the presence of structurally intact ADSe at the active site. Electrochemical assays reveal that the selenium containing enzymes are more biased towards hydrogen production than their native counterparts. In contrast to previous chalcogenide exchange studies, the S to Se exchange herein is not based on a simple reconstitution approach using ionic cluster constituents but on the in vitro maturation with a pre-synthesized selenium-containing [2Fe] mimic. The combination of biological and chemical methods allowed for the creation of a novel [FeFe]-hydrogenase with a [2Fe2Se]-active site which confers individual catalytic features.

A structural view of synthetic cofactor integration into [FeFe]-hydrogenases.

[FeFe]-hydrogenases are nature's fastest catalysts for the evolution or oxidation of hydrogen. Numerous synthetic model complexes for the [2Fe] subcluster (2FeH) of their active site are known, but so far none of these could compete with the enzymes. The complex Fe2[µ-(SCH2)2X](CN)2(CO)42- with X = NH was shown to integrate into the apo-form of [FeFe]-hydrogenases to yield a fully active enzyme. Here we report the first crystal structures of the apo-form of the bacterial [FeFe]-hydrogenase CpI from Clostridium pasteurianum at 1.60 Å and the active semisynthetic enzyme, CpIADT, at 1.63 Å. The structures illustrate the significant changes in ligand coordination upon integration and activation of the [2Fe] complex. These changes are induced by a rigid 2FeH cavity as revealed by the structure of apoCpI, which is remarkably similar to CpIADT. Additionally we present the high resolution crystal structures of the semisynthetic bacterial [FeFe]-hydrogenases CpIPDT (X = CH2), CpIODT (X = O) and CpISDT (X = S) with changes in the headgroup of the dithiolate bridge in the 2FeH cofactor. The structures of these inactive enzymes demonstrate that the 2FeH-subcluster and its protein environment remain largely unchanged when compared to the active enzyme CpIADT. As the active site shows an open coordination site in all structures, the absence of catalytic activity is probably not caused by steric obstruction. This demonstrates that the chemical properties of the dithiolate bridge are essential for enzyme activity.

Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution.

[FeFe] hydrogenases are biocatalytic model systems for the exploitation and investigation of catalytic hydrogen evolution. Here, we used vibrational spectroscopic techniques to characterize, in detail, redox transformations of the [FeFe] and [4Fe4S] sub-sites of the catalytic centre (H-cluster) in a monomeric [FeFe] hydrogenase. Through the application of low-temperature resonance Raman spectroscopy, we discovered a novel metastable intermediate that is characterized by an oxidized [FeIFeII] centre and a reduced [4Fe4S]1+ cluster. Based on this unusual configuration, this species is assigned to the first, deprotonated H-cluster intermediate of the [FeFe] hydrogenase catalytic cycle. Providing insights into the sequence of initial reaction steps, the identification of this species represents a key finding towards the mechanistic understanding of biological hydrogen evolution.

Biomimetic assembly and activation of [FeFe]-hydrogenases.

Hydrogenases are the most active molecular catalysts for hydrogen production and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN(-) ligands. Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.

The exceptional photofermentative hydrogen metabolism of the green alga Chlamydomonas reinhardtii.

The photosynthetic green alga Chlamydomonas reinhardtii is capable of performing a complex fermentative metabolism which is related to the mixed acid fermentation of bacteria such as Escherichia coli. The fermentative pattern includes the products formate, ethanol, acetate, glycerol, lactate, carbon dioxide and molecular hydrogen (H(2)). H(2) production is catalysed by an active [Fe]-hydrogenase (HydA) which is coupled with the photosynthetic electron-transport chain. The most important enzyme of the classic fermentation pathway is pyruvate formate-lyase, which is common in bacteria but seldom found in eukaryotes. An interaction between fermentation, photosynthesis and H(2) evolution allows the algae to overcome long periods of anaerobiosis. In the absence of sulphur, the cells establish a photofermentative metabolism and accumulate large amounts of H(2).

Hydrogen production. Green algae as a source of energy.

Hydrogen gas is thought to be the ideal fuel for a world in which air pollution has been alleviated, global warming has been arrested, and the environment has been protected in an economically sustainable manner. Hydrogen and electricity could team to provide attractive options in transportation and power generation. Interconversion between these two forms of energy suggests on-site utilization of hydrogen to generate electricity, with the electrical power grid serving in energy transportation, distribution utilization, and hydrogen regeneration as needed. A challenging problem in establishing H(2) as a source of energy for the future is the renewable and environmentally friendly generation of large quantities of H(2) gas. Thus, processes that are presently conceptual in nature, or at a developmental stage in the laboratory, need to be encouraged, tested for feasibility, and otherwise applied toward commercialization.

A novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain.

Hydrogen evolution is observed in the green alga Scenedesmus obliquus after a phase of anaerobic adaptation. In this study we report the biochemical and genetical characterization of a new type of iron hydrogenase (HydA) in this photosynthetic organism. The monomeric enzyme has a molecular mass of 44.5 kDa. The complete hydA cDNA of 2609 base pairs comprises an open reading frame encoding a polypeptide of 448 amino acids. The protein contains a short transit peptide that routes the nucleus encoded hydrogenase to the chloroplast. Antibodies raised against the iron hydrogenase from Chlamydomonas reinhardtii react with both the isolated and in Escherichia coli overexpressed protein of S. obliquus as shown by Western blotting. By analyzing 5 kilobases of the genomic DNA, the transcription initiation site and five introns within hydA were revealed. Northern experiments suggest that hydA transcription is induced during anaerobic incubation. Alignments of S. obliquus HydA with known iron hydrogenases and sequencing of the N terminus of the purified protein confirm that HydA belongs to the class of iron hydrogenases. The C terminus of the enzyme including the catalytic site (H cluster) reveals a high degree of identity to iron hydrogenases. However, the lack of additional Fe-S clusters in the N-terminal domain indicates a novel pathway of electron transfer. Inhibitor experiments show that the ferredoxin PetF functions as natural electron donor linking the enzyme to the photosynthetic electron transport chain. PetF probably binds to the hydrogenase through electrostatic interactions.

Transcriptional and mutational analysis of the uptake hydrogenase of the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

A 10-kb DNA region of the cyanobacterium Anabaena variabilis ATCC 29413 containing the structural genes of the uptake hydrogenase (hupSL) was cloned and sequenced. In contrast to the hupL gene of Anabaena sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the hupL gene during the heterocyst differentiation of A. variabilis. The hupSL genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5' start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The hupSL operon in A. variabilis was interrupted by an interposon deletion (mutant strain AVM13). Under N(2)-fixing conditions, the mutant strain exhibited significantly increased rates in H(2) accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H(2) developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type.

Induction, localization and metal content of hydrogenase in the green alga Chlamydomonas reinhardtii.

The hydrogenase enzyme occurring in Chlamydomonas reinhardtii is induced by anaerobic adaptation of the cells. In aerobically growing cells, antibodies against the hydrogenase failed to detect either active or inactive enzyme. However, already 10 min after the onset of anaerobic adaptation, the protein could be detected. The maximal amount of enzyme was reached after 2-3 hours anaerobiosis. Addition of nickel or iron to the growth medium did not influence activity. In atomic absorption experiments, a Ni/Fe ratio of about 1:250 was measured. We, therefore, propose the hydrogenase from C. reinhardtii to be of the Fe-only type. Adaptation in the presence of uncouplers of phosphorylation showed this process to be energy-dependent. From protein synthesis inhibition experiments, it is concluded that the protein is synthesized on cytoplasmic ribosomes and, therefore, must be nuclear encoded. After isolation of intact chloroplasts from adapted cells, the active enzyme was shown, by Western-blotting analysis, to be located in the chloroplasts.

Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii.

Hydrogenase from Chlamydomonas reinhardtii was purified to homogeneity by five column-chromatography steps under strict anaerobic conditions. The cells were disrupted by mild treatment with detergent. The enzyme was purified 6100-fold, resulting in a specific activity for H2 evolution of 935 mumol.min-1.mg protein-1 at 25 degrees C, using reduced methyl viologen as electron donor. The optimal temperature for hydrogen evolution is 60 degrees C, the optimal pH value is 6.9. The Km value for methyl viologen is 0.83 mM, for ferredoxin, 35 microM. From SDS/PAGE gels, the protein was judged to be pure. On non-denaturing gels, run under nitrogen, a single band was detected after activity staining. This band corresponded to the single band observed on denaturing SDS gels, which had an apparent molecular mass of 48 kDa. If the band was cut out of the native gel and incubated with reduced methyl viologen, hydrogen evolution could be measured. The purified enzyme contains 4 Fe atoms/mol. The amino acid composition and the N-terminal amino acid sequence (24 residues) of the protein were determined. No significant amino acid sequence homologies could be found to any sequences from prokaryotic hydrogenases.

[Arthrolysis of the elbow in posttraumatic contracture]. / Die Ellbogenarthrolyse bei posttraumatischer Gelenksteife.

Intra- and periarticular fractures about the elbow joint are treated with open reduction and internal fixation. This allows early functional after-treatment. Nevertheless, the range of motion remains more or less unsatisfactory. In these cases open arthrolysis provides a considerable improvement in joint function. We therefore recommend this operation when the hardware is removed about 9 months after the accident. The reasons for post-traumatic contracture of the elbow could be intrinsic such as interposed fragments, intra-articular adhesions, incongruity of the articular surfaces--or extrinsic--like contractures of the capsule and ligaments, adhesions of different layers, ectopic bone formations. In most cases a combination of both can be found. Important conditions for successful arthrolysis are mostly intact joint surfaces, failure of all conservative efforts to improve the arc of motion, a motivated patient who understands clearly the risks and benefits that could reasonably be expected by the operative procedure and rehabilitation and, last but not least, a skilled, experienced surgeon. The choice of the approach depends on the main location of the post-traumatic changes and on previous incisions. Osteotomy of the radial epicondyle gives a much better view of the joint and should be performed whenever necessary. The exact course of the operation may not be standardized. The main point is to remove scarred adhesions and bony irregularities. An individually modified rehabilitation program is as important as the operative procedure itself to achieve the best results possible. In general, the exercises should not cause pain. In the first few days plaster casts in flexion and extension are used. Physiotherapy is supported by CPM machines as early as possible. Patients must be prepared with the help of drugs and the application of ice bags. Even after months improvement of motion can be obtained. In a retrospective follow-up study, 125 out of 168 patients with arthrolysis of the elbow joint were reviewed. Most patients sustained a fracture of the distal humerus. In 77%, the results were graded as very good, good or satisfactory, i.e., the average relative improvement amounted to at least 40% according to the criteria of W. Blauth. Patients with very severe (preoperative ROM 0-30 degrees) and severe (preoperative ROM 30-60 degrees) contractures profited more (relative improvement 60%) than the others (relative improvement 45%). Overall, the average arc of total motion increased 49 degrees; the relative improvement of motion increased by 58%.

Glycosylated hemoglobin in patients treated by chronic dialysis.

A glycosylated hemoglobin (HbA1) test was used to evaluate the role of dialysate glucose in the development of carbohydrate intolerance and hyperlipidemia in chronic hemodialysis patients and chronic peritoneal dialysis patients. HbA1 levels were significantly elevated in all groups of patients. HbA1 levels were not ameliorated with 8 weeks of glucose-free hemodialysis. There was no correlation between HbA1 and serum glucose, triglycerides, or cholesterol. Thus, HbA1 elevation cannot be explained solely by glucose reabsorption from dialysate. This test is helpful in the detection of carbohydrate intolerance, but its usefulness in evaluation of hyperlipidemia of dialysis patients is uncertain.